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| United States Patent |
5,151,372
|
|
Kuroiwa
, et al.
|
September 29, 1992
|
Method for determination of hydroxyacetophenone derivatives
Abstract
There is provided a method for determination of hydroxyacetophenone
derivatives such as 3,5-dichloro-4-hydroxyacetophenone in the presence of
protein such as albumins or globulins with high sensitivity. The
hydroxyacetophenone derivative can be measured in the neutral to acidic
region, whereby the method is not affected by interferrants such as
bilirubin or hemoglobin. The hydroxyacetophenone derivatives are useful as
the chromogen of synthetic substrates for determining acid phosphatase
activity.
| Inventors:
|
Kuroiwa; Katsumasa (Koriyama, JP);
Katayama; Katsuhiro (Koriyama, JP);
Miura; Toshihide (Koriyama, JP);
Nagasawa; Takeshi (Urawa, JP)
|
| Assignee:
|
Nitto Boseki Co., Ltd. (Fukushima, JP)
|
| Appl. No.:
|
483903 |
| Filed:
|
February 22, 1990 |
Foreign Application Priority Data
| Current U.S. Class: |
436/164; 436/8; 436/88; 436/128; 436/131; 436/171 |
| Intern'l Class: |
G01N 021/00; G01N 031/00; G01N 033/00 |
| Field of Search: |
436/88,8,131,128,171,164
|
References Cited
U.S. Patent Documents
| 5081274 | Jan., 1992 | Kuroiwa et al. | 358/198.
|
Other References
Chem. Abst. 114:159857d, 1991.
Chem. Abst. 94:184914w, 1981.
Chem. Abst. 92:140047v, 1980.
|
Primary Examiner: Rollins; John W.
Attorney, Agent or Firm: Darby & Darby
Claims
What is claimed is:
1. A method for the determination of a hydroxyacetophenone derivative which
comprises measuring a UV absorbance of said derivative, in association
with a protein, said derivative represented by formula (I):
##STR4##
wherein R represents --(CH.sub.2).sub.n CH.sub.3 (n=0 to 3); and X.sub.1
and X.sub.2 independently represents a halogen atom or hydrogen atom.
2. A method according to claim 1, wherein said hydroxyacetophenone
derivative is 3,5-dichloro-4-hydroxyacetophenone.
3. A method according to claim 1, wherein said protein is selected from the
group consisting of albumin and globulin.
4. A method according to claim 3, wherein said albumin is selected from the
group consisting of human serum albumin and bovine serum albumin.
5. A method according to claim 3, wherein said globulin is
.gamma.-globulin.
6. A method according to claim 1, wherein said absorbance is measured at a
wavelength from 300 to 370 nm.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for determination of
hydroxyacetophenone derivatives. More particularly, the present invention
relates to a method for determination of hydroxyacetophenone derivatives
which comprises measuring hydroxyacetophenone derivatives represented by
general formula (I):
##STR1##
wherein R represents --(CH.sub.2).sub.n CH.sub.3 (n=0 to 3); and X.sub.1
and X.sub.2 independently represents a halogen atom or hydrogen atom, in
association with protein. The present invention also relates to a method
for determination of a hydrolase activity utilizing such a measurement
method. Hydroxyacetophenone derivatives are useful as the chromogen of
synthetic substrates for determining a hydrolase activity. Therefore, by
applying the methods described above, the activity of hydrolases such as
acid phosphatase or the like can be determined with high sensitivity.
2. Prior Art Statement
In general, hydroxyacetophenone derivatives can be determined by measuring
their absorbance 340 nm with a spectrophotometer since the maximum
absorption of their absorption spectra exists at about 340 nm.
Hydroxyacetophenone derivatives have a structure containing a hydroxy
group therein. By utilizing the hydroxy moiety for ester bond or ether
bond, the derivatives are expected to be promising as the chromogen of
synthetic substrates for determining the hydrolase activity, which are
used for clinical inspections.
In recent clinical inspections, the main trend is by means of an automated
analyzer. For this reason, synthetic substrates available to analysis of
an initial velocity are desired also in activity measurement of enzyme
adoptable to automated analysis. With respect to hydrolases such as acid
phosphatase, N-acetylglucosaminidase, .beta.-galactosidase,
.alpha.-amylase, etc. which have the optimum pH in the range of neutral to
acidic regions, however, there are known few chromogens that can be
colorimetrically determined in the rang of neutral to acidic regions so
that the initial velocity method has not spread very widely. Methods for
determination of the activity of these enzymes using conventional
synthetic substrates are roughly classified into the following 3 types, in
which chromogens used are also illustratively given.
(a) Method which comprises using p-nitrohenylphosphoric acid as substrate,
adding an alkali to the chromogenic p-nitrophenol hydrolyzed and released
upon enzyme reaction and colorimetrically determining the chromogen
quantitatively [Hudson, P. B.: J. Urol., 58, 89 (1947)]
(b) Method which comprises using phenylphosphoric acid as substrate,
condensing the similarly released chromogenic phenol with other compounds
and colorimetrically determining the chromogen quantitatively [Kind, P. R.
N., King, E. J.: J. Clin. Path., 7, 322 (1954)]
(c) Method which comprises using 2,6-dichloro-4-nitrophenylphosphoric acid
as substrate and quantitatively determining the chromogenic
2,6-dichloro-4-nitrophenol colorimetrically as it is [Teshima, S.,
Hayashi, Y., Ando, M.: Clin. Chim. Acta, 168, 231 (1987)]
However, in the methods (a) and (b) described above, a color-forming
reaction for termination and condensation are required, respectively so
that it is impossible to conduct a rate assay. In the method (c), it is
possible to perform the initial velocity analysis. However, substrates
obtained using currently found chromogens involve problems that they tend
to cause spontaneous hydrolysis and are unstable and furthermore,
2,6-dichloro-4-nitrophenol as the chromogen is measured at about 400 nm
and the substrates are thus liable to be affected by the coexisting
substances such as bilirubin or hemoglobin in body fluids such as serum or
urine.
In this regard, the hydroxyacetophenone derivatives can be expected to as
the chromogen of substrates for determination of hydrolases described
above, since their wavelength for measurement can be set at about 340 nm
which is affected only with difficulty by the coexisting substances.
However, use of the hydroxyacetophenone derivatives alone does not result
in sufficiently large absorption at 340 nm. In addition, dissociation of
the hydroxy group is also insufficient in the acidic region. Thus, the
hydroxyacetophenone derivatives encounter problems that their sensitivity
for measurement is poor and the initial velocity method does not
satisfactorily work in the acidic region. Such problems remain unsolved
yet.
In order to solve the foregoing problems, the present inventors have made
extensive investigations and have reached the present invention. That is,
the present inventors have made investigations of the use
hydroxyacetophenone derivatives represented by general formula (I)
described below, in association with protein, in the UV measurement method
at about 340 nm. As a result, they have found that there is an increase in
the absorption peak at about 340 nm, i.e., an increase in molecular
extinction coefficient .epsilon. and acceleration of dissociation of the
hydroxy group at the acidic region, i.e., a shift of pKa showing a pH
value in 50% dissociation of the hydroxy group in the hydroxyacetophenone
derivatives toward the acidic region. They have also found that in this
case, the measurement sensitivity can be increased, it becomes possible to
perform the measurement at the acidic region, and even in the case of
using as the chromogen of substrates for determination of hydrolase
activity, the measurement sensitivity can be increased, it becomes
possible to perform the initial velocity method at the acidic region.
SUMMARY OF THE INVENTION
That is, the present invention relates to a method for determination of
hydroxyacetophenone derivatives which comprises measuring
hydroxyacetophenone derivatives represented by general formula (I):
##STR2##
wherein R represents --(CH.sub.2).sub.n CH.sub.3 (n=0 to 3); and X.sub.1
and X.sub.2 independently represents a halogen atom or hydrogen atom], in
association with protein. The present invention also relates to a method
for determination of a hydrolase activity utilizing such a measurement
method. It is thus an object of the present invention to provide a method
for determination of the hydroxyacetophenone derivatives which enables to
measurement in the acidic region with high sensitivity.
Another object of the present invention is to provide a method for
determination of the hydroxyacetophenone derivatives without being
affected by bilirubin, hemoglobin, etc. in body fluids.
A further object of the present invention is to provide a method for
determination of the hydroxyacetophenone derivatives which is applicable
to the initial velocity method.
These and other objects and advantages will be apparent from the following
description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a dissociation curve of 3,5-dichloro-4-hydroxyacetophenone.
FIG. 2 is a graph showing a standard curve for
3,5-dichloro-4-hydroxy-acetophenone.
FIG. 3 indicates a reaction time course of
2,6-dichloro-4-acetylphenylphosphoric acid.
DETAILED DESCRIPTION OF THE INVENTION
In the formula (I) described above, examples of R include methyl, ethyl,
propyl and butyl. X.sub.1 and X.sub.2 independently represents a halogen
exemplified by fluorine, chlorine, bromine, etc.
The compounds represented by general formula (I) can be readily obtained,
for example, by Fries rearrangement of the corresponding phenol ester
compounds (Japanese Patent Application No. 63-234555).
The protein which can be used in association with the compounds of formula
(I) may be any protein so long as it is water soluble. Examples of such
protein include albumins such as human serum albumin, bovine serum
albumin, etc.; globulins such as .gamma.-globulin, etc. A concentration of
these proteins is not particularly limited but it may be sufficient that
the protein is contained preferably in a concentration of approximately
0.1 to 1% in a buffer solution used in performing the method of the
present invention.
The term "in association with protein" as used herein refers to the system
in which protein is allowed to coexist in the measurement system. More
specifically, the protein may be mixed with the hydroxyacetophenone
derivatives of formula (I); may be dissolved in a buffer solution used in
the measurement system and the solution is mixed with the compounds of
formula (I). In any event, it is sufficient to attain the effects of the
present invention whatsoever the protein is present in the measurement
system, in combination with the hydroxyacetophenone derivatives of formula
(I).
As the buffer solution, any conventional buffer solution is usable as long
as the solution has a buffering ability in the range of from the neutral
to acidic regions. Examples of the buffer solution include buffer
solutions of citric acid, acetic acid, succinic acid, phthalic acid,
phosphoric acid, boric acid, etc. If necessary and desired, a surface
active agent, a preservative, a stabilizer and the like may also be added
to these buffer solutions.
Changes in .epsilon. at 340 nm (.epsilon..sub.340) and pKa of the
hydroxyaetophenone derivatives in the presence of or absence of protein
were examined The results are shown below.
EXPERIMENT 1
A buffer solution containing 0.0286M citric acid, KH.sub.2 PO.sub.4, boric
acid, diethylbarbituric acid and 0.1% Triton X-100, and a buffer solution
further supplemented with 0.3% bovine serum albumin (hereafter simply
referred to as BSA, manufactured by Sigma, Inc.) were adjusted to pH
ranges of from 3 to 10. To 0.38 ml of each of these buffer solutions, was
added 0.02 ml of each of the hydroxyacetophenone derivatives shown in
Table 1 below, which had been adjusted to 0.1 mM. Using a
spectrophotometer, absorbance of each solution was measured at 340 nm.
In this case, .epsilon..sub.340 and pKa of each of the hydroxyacetophenone
derivatives in the presence or absence of BSA are shown in Table 1. In
addition, changes in absorbance of 3,5-dichloro-4-hydroxyacetophenone at
various pH values and its pKa are shown in FIG. 1 (absorbance of the
buffer solution itself is removed from the absorbance shown).
TABLE 1
______________________________________
##STR3## BSA, 0.3%(pH 6.0)cm.sup.-1) .times. 10.sup.-3.epsilon..sub.
40 (l .multidot. mol.sup.-1 .multidot.
BSA, 0.3%pKa
______________________________________
R X.sub.1 X.sub.2 None Added None Added
______________________________________
CH.sub.3
F F 12.7 14.3 5.13 4.94
CH.sub.3
Cl Cl 14.9 22.9 4.56 4.19
CH.sub.3
Br Br 16.7 24.2 4.50 4.02
CH.sub.3
Cl H 4.9 6.9 6.43 6.03
CH.sub.3
Br H 5.3 9.7 6.37 5.81
C.sub.3 H.sub.7
Cl Cl 15.0 16.7 4.89 4.00
______________________________________
As is clear from these results, the hydroxyacetophenone derivatives can
increase .epsilon..sub.340 by about 10% to about 80% in the presence of
BSA. In addition, pKa is also shifed to the acidic region by approximately
0.2 to 0.9. Therefore, according to the method of the present invention,
it is possible to determine the hydroxyacetophenone derivatives in the
acidic region with higher sensitivity than in conventional methods.
According to the present invention, the determination of the
hydroxyacetophenone derivatives represented by general formula (I) can be
generally carried out as follows. That is, after a solution of the
hydroxyacetophenone derivative to be determined is mixed with a buffer
solution containing a suitable protein, its absorbance is measured at 300
to 370 nm, preferably at the maximum wavelength of from 330 to 340 nm,
using a spectrophotometer. An amount of the hydroxyacetophenone derivative
to be measured is determined by comparing the standard curve preliminarily
obtained by similarly performing with respect to known amounts of the
hydroxyacetophenone derivative.
The measurement method of the present invention is applicable to
determination of the activity of hydrolases having the optimum pH in the
range of neutral to acidic regions, such as acidic phosphatase,
N-acetylglucosamidase, .beta.-galactosidase, .alpha.-amylase, etc., using,
e.g., the initial velocity method. That is, when synthetic substrates for
determining such enzymes using the hydroxyacetophenone derivative as the
chromogen and the enzyme to be determined are subjected to enzyme reaction
and the protein is present in the measurement system, the hydroxy group
released from the hydroxyacetophenone derivative as the chromogen upon the
enzyme reaction is sufficiently dissociated even in the range of neutral
to acidic regions and .epsilon. also increases. By measuring an increase
of the absorbance per one minute, the activity of the enzyme can be
determined by the initial velocity method with high sensitivity. As the
synthetic substrate for determining the hydrolase activity in which the
hydroxyacetophenone derivative is used as the chromogen, there are
phosphoric acid derivatives such as 2,6-dichloro-4-acetylphenylphosphoric
acid, etc. obtained by binding, e.g., hydroxyacetophenone derivatives with
phosphoric acid through ester bond (Japanese Patent Application No.
63-234555). By using the phosphoric acid derivatives as substrate,
especially acid phosphatase activity can be determined with high
sensitivity.
According to the present invention, the problems encountered in
conventional methods have been solved in various points. The advantages of
the present invention are given below.
(1) The present invention provides the following advantages by using the
hydroxyacetophenone derivative as the chromogen of synthetic substrates
for determining hydrolase activity which have the optimum pH in the
neutral to acidic regions and by determining the hydrolase activity in the
co-presence of protein in the measurement system.
(a) Even in the acidic region, the hydroxy group of the hydroxyacetophenone
derivative is sufficiently dissociated so that it becomes possible to
measure the hydrolase activity by the initial velocity method. Thus, an
automated analyzer is applicable to the determination of hydrolase
activity.
(b) Due to increased .epsilon. of the hydroxyacetophenone derivative, the
measurement sensitivity of the enzyme activity increases.
(c) The wavelength for measurement can be set in the UV region of from 300
to 370 nm and hence, the method is affected only with difficulty, upon
measurement, by coexisting substances such as bilirubin, hemoglobin, etc.
in body fluids.
(2) In the measurement of the hydroxyacetophenone derivative, the following
advantages are given by allowing protein to coexist.
(a) Since pKa of the hydroxyacetophenone derivative is shifted to the
acidic region, it has become possible to measure the hydroxyacetophenone
derivative in an acidic solution, which was impossible before.
(b) Because of increased .epsilon. of the hydroxyacetophenone derivative,
the measurement sensitivity increases.
As described above, the method for measurement of the present invention not
only provides an improvement in determining the hydroxyacetophenone
derivative but also enables to perform the initial velocity analysis for
determination of the hydrolase activity having the optimum pH in the
neutral to acidic regions which leads to application of the present method
to an automated analyzer. Therefore, the present invention greatly
contributes to the field of clinical tests.
EXAMPLES
Hereafter the present invention is described in more detail with reference
to the examples below but is not deemed to be limited thereto.
EXAMPLE 1
Standard curve of the hydroxyacetophenone derivative
As a stock solution (5/5), 0.2 mM solution of
3,5-dichloro-4-hydroxyacetophenone was used. The solution was diluted with
purified water to prepare 5-stage serial dilutions. To 0.02 ml of the
solution in each concentration was mixed with 0.38 ml each of 0.1M citrate
buffer solution (pH 5.4) containing 0.1% Triton X-100 and a buffer
solution further supplemented with 0.3% BSA. Each absorbance was measured
at 340 nm with a spectrophotometer to prepare the standard curve (FIG. 2).
As is clear from the drawing, when BSA is present, the absorbance is
extremely large Therefore, as compared to the case where BSA is absent,
the measurement sensitivity is greatly improved. Since the graph shows the
proportional relationship up to 5/5, it is possible to quantitatively
determine 3,5-dichloro-4-hydroxyacetophenone up to 0.01 mM.
EXAMPLE 2
Determination of Acid Phosphatase Using Substrate Obtained by Using the
Hydroxyacetophenone Derivative as the Chromogen
The activity of acid phosphatase in a vital sample solution was determined
using 2,6-dichloro-4-acetylphenylphosphoric acid, which was obtained using
3,5-dichloro-4-hydroxyacetophenone as the chromogen. The substrate was
synthesized according to the method described in Japanese Patent
Application No. 63-234555 The outline of the measurement is described
below.
In 0.01M citrate buffer solution (pH 3.0) was dissolved
2,6-dichloro-4-acetylphenylphosphoric acid to make 6 mM substrate solution
A sample (prostatic acid phosphatase; manufactured by Sigma, Inc.; 0.02
ml) was added to 0.4 ml each of 0.1M citrate buffer solution (pH 5.4)
containing 0.1% Triton X-100 and a buffer solution further supplemented
with 0.3% BSA. After warming at 37.degree. C. for 3 minutes, 0.1 ml of the
substrate solution was added thereto to initiate the reaction A change in
absorbance was measured at 340 nm and the change in absorbance per one
minute was determined (for the measurement, Model No. 7050 automated
analyzer manufactured by Hitachi Ltd. was used).
The change in absorbance per one minute (.DELTA.E/min) and data (blank)
obtained using physiological saline as a sample solution are shown in
Table 2. The time course for 5 minutes is shown in FIG. 3.
TABLE 2
______________________________________
Sample
Presence or Prostatic Acid
Physiological
Absence of Phosphatase Saline (blank)
0.3% BSA *(.DELTA.E/min)
(.DELTA.E/min)
______________________________________
None 0.0864 0.0012
Presence 0.1345 0.0029
______________________________________
*(Blank value is compensated)
As is obviously noted from the results, .DELTA.E/min becomes about 1.6
times by the addition of BSA so that the measurement sensitivity
increases. Since an increase in the blank value is small, the substrate is
stable. Furthermore, since the time course for 5 minutes is in the linear
relationship, the acid phosphatase activity can be determined by the
initial velocity method, using the substrate in which the
hydroxyacetophenone derivative of the present invention is used as the
chromogen, in association with protein.
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